Physiologically active substances of plant, process for the preparation thereof, and utilities thereof

ABSTRACT

The present invention relates to compounds represented by the following formula ##STR1## wherein R 1 , R 2 , R 3 , R 4 , and R 5  independently represent a hydrogen atom or an acetyl or benzyl group, R 6  represents a hydrogen atom a hydroxyl, acetoxy, or benzyloxy group, R 7  represents a hydrogen atom or R 6  and R 7  together my represent another direct bond, and R 8  represents a carboxyl, methoxycarbonyl, hydroxymethyl, or acetoxymethyl group or salts thereof and a process for the preparation thereof, as well as their utilities in a method for culture, a method for the growth inhibition of moyashi roots, and a method for the growth promotion of moyashi hypocotyls.

INTRODUCTION

The present invention relates to physiologically active substances ofplant with a novel structure being useful for the growth regulation ofagricultural products etc., a process for the preparation thereof, andutilities thereof.

BACKGROUND OF THE INVENTION

At present, "allelopathy", which is represented by the phenomenon ofpromotion of ripening fruits by ethylene released from other ripenedfruits, is known and observed in every plant including microorganisms.Nowadays, such a phenomenon is attempted to be applied positively to thegrowth regulation of plants (H. Molisch: "Der Einfluss einer Pflanze aufdie andere Allelopathie", Gustav Fischer Verlag, Jena, 1937), and ispractically applied in some cases (E. L. Rice (1984): Allelopathiceffects of crop plants on other crop plants. In "Allelopathy" 2rid ed.pp. 41-67, Academic press, Inc.).

As "allelopathy", it was found that if specific kinds of seeds orseedlings are cultured in the presence of cress plant seeds in a Petridish, then the growth of hypocotyls is promoted, whereas the growth ofseedling roots is inhibited (Zasso Kenkyu, 37, 68 (1992); Zasso Kenkyu,37, 71 (1992)).

However, the substantiality of substance bringing about such"allelopathy" has still not been identified.

"Hydroponics", i.e. a method of growing plants in an aqueous solutionwith nutrients dissolved, was carried out widely as "water culture" inthe old days, and has become a method more frequently adopted owing tothe development in plant biotechnology in recent years.

The most important item for carrying out hydroponics is to select asuitable composition to be used for a culture solution.

Typical examples of such culture solutions are Sachs' solution, Knop'ssolution, Hoagland's solution, etc.

Under the circumstances, the development of hydroponics using a newculture solution has been desired with the aim of establishing moreefficient hydroponics.

Bean sprouts, barley malt, etc., are mainly grown legume sprouts in thedark (referred to as "moyashi", hereinafter) and 1 week or so isrequired for their industrial production. In recent years, it has beenconducted to inhibit the growth of roots and remove roots mechanicallyin the production of moyashi in order to improve their value ascommercial products.

It has therefore been desired to develop a method for producing moyashiof enhanced value as a commercial product in higher productivity.

In order to solve the above problem, the present inventors have isolatedand identified, from seedlings of cress etc., physiologically activesubstances bringing about "allelopathy", have succeeded in the synthesisof said substances and derivatives thereof, and have established a newculture method comprising use of a new artificial soil or culturesolution, and they have found that said substances can promote thegrowth of moyashi simultaneously with the growth inhibition of roots,thereby arriving at the completion of the present invention.

SUMMARY OF THE INVENTION

The present invention comprises the following inventions.

1. Compounds represented by the following formula (I): ##STR2## whereinR¹, R², R³, R⁴, and R⁵ independently represent a hydrogen atom, or anacetyl or benzyl group, R⁶ represents a hydrogen atom, a hydroxyl,acetoxy, or benzyloxy group, R⁷ represents a hydrogen atom, or R⁶ and R⁷together may represent another direct bond, and R⁸ represents acarboxyl, methoxycarbonyl, hydroxymethyl, or acetoxymethyl group, andsalts thereof.

2. Compound represented by the following formula (Ia): ##STR3## andsalts thereof. 3. Compounds represented by the following formula (Ib):##STR4## wherein R¹ ', R² ', R³ ', R⁴ ', and R⁵ ' independentlyrepresent a hydrogen atom, or an acetyl or benzyl group, R⁶ ' representsa hydrogen atom, a hydroxyl, acetoxy, or benzyloxy group, R⁷ 'represents a hydrogen atom, or R⁶ ' and R⁷ ' together may representanother direct bond, R⁸ ' represents a carboxyl, methoxycarbonyl,hydroxymethyl, or acetoxymethyl group, in which, if R¹ ', R² ', R³ ', R⁴', and R⁵ ' each represent a hydrogen atom, and R⁸ ' represents acarboxyl group, then R⁶ ' represents a hydrogen atom, a hydroxyl,acetoxy, or benzyloxy group and R⁷ ' represents a hydrogen atom, andsalts thereof.

4. Compounds as defined in claim 1, wherein R¹, R², R³, R⁴, and R⁵ eachrepresent a hydrogen atom, R⁶ represents a hydroxyl group, R⁷ representsa hydrogen atom, and R⁸ represents a carboxyl group, and salts thereof.

5. Compounds as defined in claim 1, wherein R¹, R², R³. R⁴, and R⁵ eachrepresent a hydrogen atom, R⁶ represents a hydroxyl group, R⁷ representsa hydrogen atom, and R⁸ represents a methoxycarbonyl group.

6. Compounds as defined in claim 1, wherein R¹, R², R³, R⁴, and R⁵ eachrepresent a hydrogen atom, R⁶ represents a hydroxyl group, R⁷ representsa hydrogen atom, and R⁸ represents a hydroxymethyl group.

7. Compounds as defined in claim 1, wherein R¹, R², R³, R⁴, and R⁵ eachrepresent a hydrogen atom, R⁶ represents a hydrogen atom, R⁷ representsa hydrogen atom, and R⁸ represents a carboxyl group, and salts thereof.

8. Compounds as defined in claim 1, wherein R¹, R², R³, R⁴, and R⁵ eachrepresent a benzyl group, R⁶ represents a benzyloxy group, R⁷ representsa hydrogen atom, and R⁸ represents an acetoxymethyl group.

9. Compounds as defined in claim 1, wherein R¹, R², R³, R⁴, and R⁵ eachrepresent an acetyl group, R⁶ and R⁷ together may represent anotherdirect bond, and R⁸ represents a methoxycarbonyl group.

10. A process for the preparation of the compound as defined in claim 2or salts thereof, which comprises hydrolysis of the compound as definedin claim 9.

11. A process for the preparation of physiologically active substancesof plant, which comprises immersing plant seeds in water and thencollecting from the extract the compound represented by formula (Ia):##STR5## or a salt thereof. 12. A process as defined in claim 11,wherein the plant seeds are those of a plant selected from the groupconsisting of slender amaranth, asparagus oat, barnyard grass, rice,green foxtail, pea, timothy grass, Persian speedwell (bird's-eyespeedwell), okra, wild oat, goose grass, turnip, pumpkin, cauliflower,cabagge, cress, cockscomb, cockspur grass, rice flat-sedge, burdock,"shikokubie", perilia, crown daisy, common purslane, celery, buckwheat,radish, "ta-inubie", small-flower umbrella-plant, cayenne pepper, corn,tomato, eggplant, leek, carrot, Chinese cabbage, parsley, sunflower,amaranth, broccoli, spinach, hardstem bulrush, matricaria, trefoil,cabgrass and lettuce.

13. A method for culture, wherein an artificial soil or a culturesolution contains compound(s) represented by the following formula (Ic):##STR6## wherein R¹ ", R² ", R³ ", R⁴ ", and R⁵ " independentlyrepresent a hydrogen atom or an acetyl group, R⁶ " represents a hydrogenatom or a hydroxyl group, R⁷ " represents a hydrogen atom, or R⁶ " andR⁷ " together may represent another direct bond, and R⁸ " represents acarboxyl, methoxycarbonyl, or hydroxymethyl group, or salt(s) thereof.

14. A method for culture, wherein an artificial soil or a culturesolution contains the compound represented by the following formula(Ia): ##STR7## or a salt thereof. 15. A method as defined in claim 14,wherein the content of the compound represented by formula (Ia) orsalt(s) thereof is from 1 to 10000 ppm.

16. A method for the growth inhibition of moyashi roots, which comprisesapplying a solution containing compound(s) represented by formula (Ic)or salt(s) thereof to moyashi seeds.

17. A method for the growth inhibition of moyashi roots, which comprisesapplying a solution containing the compound represented by formula (Ia)or salt(s) thereof to moyashi seeds.

18. A method for the growth promotion of moyashi hypocotyls, whichcomprises applying a solution containing compound(s) represented byformula (Ic) or salt(s) thereof to moyashi seeds.

19. A method for the growth promotion of moyashi hypocotyls, whichcomprises applying a solution containing the compound represented byformula (Ia) or salt(s) thereof to moyashi seeds.

According to the present invention, there are provided physiologicallyactive substances with a new structure useful for the growth regulationof agricultural products etc., a novel culture method effective for thegrowth of plants, and a method for the industrial production of moyashiof enhanced value as a commercial product in high productivity.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of a sodium salt of compound (Ia) on the growthof hypocotyls (part of a plant above the ground) and roots. FIG. 1 alsoshows the results of gibberellin (GA₃) and indoleacetic acid (IAA).

FIG. 2 is a photograph of plants whose growth was promoted by thepresent compound obtained by extraction of sunflower seeds.

A shows Amaranthus caudatus L.

B shows Lettuce.

C shows Persian speedwell.

D shows timothy.

FIG. 3 shows the results of plant growth promotion on 20 kinds of plantseeds by the present compound obtained by extraction of sunflower seeds.

FIG. 4 is a photograph of lettuces after hydroponics using the presentcompound. FIG. 4 shows lettuces cultured in water only (control) andwater containing the present compound in an amount derived from 50, 100,150, and 200 sunflower seeds (from left to right).

FIG. 5 is a photograph of peas after hydroponics using the presentcompound. FIG. 5 shows peas cultured in water only (control) and watercontaining the present compound in an amount derived from 50, 100 and150 sunflower seeds (from left to right).

FIG. 6 is a photograph of tomatoes after hydroponics using the presentcompound. FIG. 6 shows tomatoes cultured in water only (control) andwater containing the present compound in an amount derived from 50, 100and 150 sunflower seeds (from left to right).

FIG. 7 is a photograph of black matpes after hydroponics using thepresent compound. FIG. 7 shows black matpes cultured in water only(control) and water containing the present compound in an amount derivedfrom 150, 500, and 1500 sunflower seeds (from left to right).

FIG. 8 is a photograph of green gram hypocotyls whose growth waspromoted by the present compound. FIG. 8 shows untreated green gram(control) at the right and green gram treated with the present compoundat the left.

FIGS. 9, 10, and 11 show IR spectrum, ¹ H-NMR spectrum, and ¹³ C-NMR ofcompound (7), respectively.

FIG. 12 shows ¹ H-NMR spectrum of compound (8a).

FIG. 13 shows ¹ H-NMR spectrum of compound (9a).

FIGS. 14, 15, and 16 show IR spectrum, ¹ H-NMR spectrum, and ¹³ C-NMRspectrum of compound (10a), respectively.

FIGS. 17, 18, and 19 show IR spectrum, ¹ H-NMR spectrum, and ¹³ C-NMRspectrum of compound (12), respectively.

FIGS. 20 and 21 show IR spectrum and ¹ H-NMR spectrum of compound (12'),respectively.

FIG. 22 shows ¹ H-NMR spectrum of compound (14).

DETAILED DESCRIPTION OF THE INVENTION

Salts of compounds represented by the above formula (I), (Ia), (Ib), or(Ic) include e.g. sodium salts and potassium salts, lithium salts.

Compounds represented by the above formula (I), (Ia), (Ib), or (Ic) orsalts thereof can be obtained by the following chemical synthesis.

As illustrated in the reaction scheme below, α-L-rhamnose is firstallowed to react with benzyl alcohol in the presence of a catalyticamount of sulfuric acid in a solvent such as benzene etc., thus givingbenzyl glycoside, which in turn is allowed to react with2,2-dimethoxypropane in a solvent such as acetone etc. in the presenceof p-toluenesulfonic acid, thereby being converted into isopropylidene(compound (2)). Compound (2) is then allowed to react with benzylbromide in the presence of sodium hydride in a solvent e.g.N,N-dimethylformamide etc., thereby giving compound (3), followed bytreatment with acetic acid-water-1,4-dioxane etc. for the elimination ofthe isopropylidene residue, so that Compound (4) is obtained.

Subsequently, compound (4) is treated with dibutyltin oxide (Bu₂ SnO) ina solvent such as benzene etc., thereby forming compound (5), which isthen treated with benzyl bromide in the presence of cesium fluoride(CsF), whereby compound (6) is obtained: ##STR8## wherein Bn representsa benzyl group, and Bu represents a n-butyl group.

As illustrated in the following reaction scheme, compound (6) is thenallowed to react compound (A) in the presence of molecular sieve andmethylsulphenyl bromide, whereby compound (7) can be obtained. Thecompound used in this step, compound (A), is a known compound which canbe synthesized in a 7-step reaction from D-glucose (Acta Chem. Scand.,43, 471 (1989); Carbohydr. Res., 202, 225 (1990)).

Compound (7) is then treated with a base e.g. potassium carbonate etc.in a solvent such as methanol etc., whereby compound (8) can beobtained. Compound (8) is catalytically reduced with palladium,palladium-carbon, palladium hydroxide, etc., thereby being convertedinto compound (8a).

Subsequently, compound (8) is treated with (1) sulfur trioxide(SO₃)-pyridine in dimethylsulfoxide (DMSO) and trimethylamine and then(2) sodium chlorite and sodium dihydrogen phosphate in t-butylalcohol-water-2-methyl-2-butene, so that compound (9) can be obtained.Compound (9) is catalytically reduced with palladium, palladium-carbon,palladium hydroxide, etc., thereby being converted into compound (9a).

Subsequently, compound (9) is treated with trimethylsilyldiazomethane ina solvent such as benzene-methanol etc., whereby compound (10) can beobtained. Compound (10) is catalytically reduced with palladium,palladium-carbon, palladium hydroxide, etc., to be converted intocompound (10a).

Subsequently, compound (10a) is acetylated with aceticanhydride-pyridine etc., thereby giving compound (11), followed bytreatment with 1,8-diazabicyclo[5.4.0.]-7-undecene (DBU) in a solvente.g. pyridine, whereby compounds (12) and (12') are formed.

Compounds (12) and (12') are then hydrolyzed with a base such as sodiumhydroxide, sodium methoxide, sodium ethoxide, sodium t-butoxide, etc.,whereby compound (1) can be obtained.

Alternatively, compounds (12) and (12') are catalytically reduced withpalladium, palladium-carbon, palladium hydroxide, etc., to be convertedinto compound (13), which in turn is hydrolyzed with a base, such assodium hydroxide, sodium methoxide, sodium t-butoxide., etc., wherebycompound (14) can be obtained. ##STR9## wherein Ac represents an acetylgroup, Ph represents a phenyl group, and Bn possesses the same meaningas defined above.

Carboxylic acids thus obtained, such as compounds (9), (9a), etc., canbe converted into the corresponding salts according to a conventionalmethod; and sodium salts, such as compounds (1), (14), etc., can beconverted into the corresponding free carboxylic acids or other saltsaccording to a conventional method.

Out of the present compounds represented by the above formula (I), thecompound wherein R¹, R², R³, R⁴, and R⁵ each represent a hydrogen atom,and R⁶ and R⁷ together represents another direct bond, and R⁸ representsa carboxyl group, i.e. the compound of the above formula (Is) or itssalt, can also be obtained by isolation and purification from cressseedlings.

In the isolation and purification of compound (Ia), cress seeds obtainede.g. from a commercial source or a natural source or by culture thereofmay be used as starting material.

Compound (Ia) can be isolated and purified from the culture solution ofcress seeds germinated in hydroponics.

Culture form in hydroponics is particularly not limited as far as cressplant seeds can be germinated.

Usually, cress plant seeds are germinated in the dark under aeration.Culture temperature is usually 15° to 30° C. preferably 20° to 25° C.Culture time is usually 1 to 2 days. The composition of a culture forhydroponics is particularly not limited, but water free from anyadditive is preferably employed for the efficient isolation andpurification of compound (Ia). Before culture, cress plant seeds arepreferably immersed in water, so that the amount of compound (Ia)secreted can be increased.

The culture solution thus obtained is concentrated in a usual manner andis then applied as necessary to gel filtration chromatography,high-performance liquid chromatography. etc., or a combination thereof,whereby compound (Ia) can be purified.

Besides the above cress plant, compound (Ia) can also be obtained byimmersing in water seeds of a plant selected from the group consistingof slender amaranth, asparagus, oat, barnyard grass, rice, greenfoxdtail, pea, timothy grass, Persian speedwell, okra, wild oat, goosegrass, turnip, pumpkin, cauliflower, cabagge, cress, cockscomb, cockspurgrass, rice flat-sedge, burdock, "shikokubie", perilla, crown daisy,common putslane, celery, buckwheat, radish, "ta-inubie", small-flowerumbrella-plant, cayenne pepper, corn, tomato, eggplant, leek, carrot,Chinese cabbage, parsley, sunflower, amaranth, broccoli, spinach,hardstem bulrush, matricaria, trefoil, cabgrass, and lettuce, followedby collecting from the extract the compound (Ia) or a salt thereof. Thesource of these seeds is not particularly limited, and it is possible toemploy seeds obtained e.g. from a commercial source or a natural sourceor by culture thereof.

Usually, cress plant seeds are germinated in the dark under aeration.Regardless of whether the seeds are to be germinated or not, temperaturefor immersion is usually 15° to 30° C., preferably 20° to 25° C.Immersion time is usually 1 to 2 days.

Tap water as well as distilled water Bay be used as water for immersion,but distilled water is preferably employed so as not to affect thephysiological activity of compound (Ia).

The immersion solution thus obtained is concentrated in a usual mannerand then applied as necessary to gel filtration chromatography,high-performance liquid chromatography, etc., or a combination thereof,whereby compound (Ia) can be purified.

Out of the present compounds thus obtained, compounds (Ic) or saltsthereof are physiologically active substances with "allelopathy" whichpromote the growth of hypocotyls of a wide variety of plants and whichpromote the growth of their roots at a low concentration whereas inhibitthe growth of their roots at a high concentration. These compounds areuseful as components in a treatment solution such as spray solution,immersion solution, etc., for use in a wide variety of culture methods,particularly for culture using an artificial soil (e.g. vermiculite),hydroponics, and a method for the growth inhibition of moyashi roots, aswell as a method for the growth promotion of moyashi hypocotyls.

For use of compound (Ic) or a salt thereof as a component in anartificial soil or culture solution, the content is usually 1 to 10,000ppm, preferably 100 ppm or more. Other effective components in theartificial soil or culture solution can be suitably selected dependingon the type of plant to be cultured. It is also possible to adoptcomponents conventionally used in an artificial soil, a culturesolution, etc.

The plants to which the present cultivation method can be applied arenot particularly limited as far as they can be cultured in an artificialsoil or culture solution. Specific examples to which the present methodis applicable are tomato, lettuce, and cabbage. However, the presentmethod is not limited to such plants, as stated above.

A desired plant can be cultured in an artificial soil or culturesolution containing compound (Ic) or a salt thereof. Culture conditionsother than those of said artificial soil or culture solution can besuitably determined depending on the type of plant to be cultured.

In case compound (Ia) (or a salt thereof) derived from above plants isused, it can be used in the form of an extract (immersion solution) assuch without isolation/purification.

In case compound (Ia) or a salt thereof is used for the growthinhibition of moyashi roots or the growth promotion of moyashihypocotyls, a solution containing 1 to 10000 ppm compound (Ic) or saltthereof is preferably sprayed or spread downwards on germinated moyashi,or otherwise germinated moyashi on the second day of culture ispreferably immersed only once in a solution containing 1 to 10000 ppmcompound (Ic) or salt thereof. This method differs from conventionalhydroponics and is characterized by spraying or sprinkling a solutioncontaining compound (Ic) or a salt thereof, or by only once immersion insaid solution for a short period of time.

For the present method for the growth inhibition of moyashi roots andfor the growth promotion of moyashi hypocotyls, compounds (Ia) or saltsthereof derived from said plants can be used in the form of an plantextract solution (immersion solution) as such withoutisolation/purification.

EXAMPLES

The present invention is further illustrated with reference to thefollowing examples, but these examples are not intended to limit thescope of the present invention.

(Example 1) Isolation and purification of the present compound fromcress seeds

(1) Preparation of plant materials

3000 cress (Lepidium sativum L.) seeds were immersed for 1 hour indeionized water.

Then, the cress seeds were placed on a stainless mesh, and thisstainless mesh was gently placed in a stainless dish (40×40×3 cm³)containing 1.6 l deionized water. The cress seeds were cultured at 25°C. for 2 days in the dark under aeration with an air pump.

(2) Purification of the present compound

1 First, the cress plant culture solution obtained in (1) above wasfiltered through Toyo No. 1 filter paper and the filtrate wasconcentrated at 85° C. under reduced pressure. Then, the concentrate waspartitioned into acetone soluble and insoluble phases.

Amaranthus caudatus L. seeds were gently placed on a filter paperimmersed with 0.8 ml of a sample solution (from said soluble orinsoluble fraction) in a Petri dish of 3 cm diameter and were thenallowed to stand at 25° C. for 5 days in the dark, and the lengths ofgerminated hypocotyls and roots were determined for the evaluation ofwhether or not any biological activity on plant growth was present inthe acetone soluble and/or insoluble phases.

As a result, the activity showing the growth promotion of hypocotyls andthe growth inhibition of roots (of Amaranthus caudatus L.) was confirmedto be present in the acetone insoluble fraction.

Subsequently, the acetone insoluble fraction was dissolved in 10 mlwater and then separated by molecular exclusion chromatography (Mol cut,Millipore Corp.) into three fractions, i.e. M_(r) above 100,000,5,000-100,000, and below 5,000. The biological activity was found to bepresent in the fraction of M_(r) below 5,000. This fraction wasconcentrated at 35° C. under reduced pressure.

2 The concentrate (approx. 150 mg) obtained in 1 above was dissolved inwater and then purified by high performance liquid chromatography (HPLC)(Waters, μ Bondasphere 5μ C. ₁₈ -100Å; column 19 mm×15 cm; eluent 100%H₂ O, flow rate 5 ml/min; 214 nm detector). The above biologicalactivity was found in fractions of retention time of 5-8 minutes.

Said HPLC fractions were combined and concentrated at 35° C. underreduced pressure and further purified by HPLC (SIC Packed Column AQ-324S-5 120A ODS; YMC Co. Ltd.; eluent 100% H₂ O, flow rate 1 ml/min; 214 nmdetector). The above biological activity was found in fractions ofretention time of 17.0-17.8 minutes. These fractions were combined andconcentrated at 35° C. under reduced pressure, whereby 6.5 mg amorphouspowder was obtained.

(3) Structural determination of the present compound

The purified sample thus obtained was analyzed for structuraldetermination. Optical rotation was determined with a JASCO A-202spectrophotometer. IR spectrum was obtained in glycerol with a JASCOA-202 spectrophotometer. UV spectrum was obtained in D₂ O with a JASCOUVIDEC-610A spectrophotometer. ¹ H-NMR spectrum was obtained with JEOLJNM-GX400 NMR spectrometer. FAB mass spectrum was recorded in glycerolmatrix.

1 Optical rotation: [α]_(D) ¹⁹ =+87.8 (c 0.032, D₂ O)

2 IR: Peaks were found at the absorption of --COOH group (1590 cm⁻¹) andOH group (3300 cm⁻¹.

3 UV: λ_(max) 225 nm (ε approx. 2,100)

4 Mass: A M⁺ +Na peak was observed at m/z 367.0591.

5 ¹ H-NMR: 65.72 (1H, d, J=3.2Hz, j), 5.17 (1H, d, J=1.6 Hz, a), 5.07(1H, d, J=2.3 Hz, g), 4.26 (1H, dd, J=6.9, 3.2 Hz, i), 4.08 (1H, dd,J=3.4, 1.6 Hz, b), 3.79 (1H, dq, J=9.7, 6.8 Hz, e), 3.76 (1H, dd, J=9.7,3.4 Hz, c), 3.72 (1H, dd, J=6.9, 2.3 Hz, h) 3.31 (1H, dd, J=0.7 Hz, d)and 1.80 (3H, d, J=6.8 Hz, f)

6 After treated overnight with acetic anydride-pyridine-MeOH at roomtemperature, the above purified sample was converted into thecorresponding methyl ester having five acetoxy groups. The result ofmass spectrum analysis indicated that the molecular formula of thisester is C₂₃ H₃₀ O₁₅ [m/z 546.1564 (M⁺)].

The IR spectrum indicated no hydroxyl absorption. The ¹ H-NMR(CDCl₃)spectrum exhibited signals for five acetoxyl methyl protons [δ2.00 (3H,s), 2.05 (3H, s), 2.10 (3H, s), 2.14 (3H×2, s] and signal for methoxylproton [δ3.88 (3H, s)].

The following results were obtained in nuclear Overhauser effect (NOE)experiments of the methyl ester. That is, irradiation at δ5.17 (H at the1-position) increased 7.3% and 8.3% the intensity of H at the 2-positionand H at the 1-position, respectively.

Irradiation at δ5.07 (H at the 1'-position) increased 8.3%, 6.9%, and13.6% the intensity of H at the 1-, 2-, and 2'-positions, respectively.

6 These results indicated that this purified sample is sodium2-O-rahmnopyranosyl-4-deoxy-threo-hex-4-enopyranosiduronate representedby the above formula (Ia), i.e. the sample proved to have the structureof the above formula (I).

(4) Determination of biological activity of the present compound Theeffect of the present compound (1) on the growth of etiolated Amaranthuscaudatus L. seedlings was examined in comparison with that ofgibberellin (GA₃) and indoleacetic acid (IAA). Determination was carriedout according to the method as described in (2) 1 above. The results areshown in FIG. 1. From the results, it was found that the presentcompound promotes the hypocotyl growth at concentrations higher than 3μM and inhibits the root growth at concentrations higher than 100 μM.The growth promotion of hypocotyls by the present compound is twenty tothirty times as much as that by gibberellin, suggesting that the presentcompound is a potent growth substance. The growth inhibition of roots bythe present compound was almost the same as that of gibberellin.Indoleacetic acid inhibited the hypocotyl and root growth.

(Example 2) Isolation and purification of the present compounds fromplant seeds other than cress

(1) Preparation of plant materials

900 seeds each of 19 kinds of plants, i.e. sunflower, spinach, corn,okra, carrot, oat, parsley, barnyard grass, cayenne pepper, buckwheat,tomato, cabbage, lettuce, cockscomb, burdock, trefoil, pea, radish, andPersian speedwell seeds were immersed for 1 hour in deionized water.Subsequently, these seeds were placed on a stainless mesh, and thisstainless mesh was gently placed in a stainless dish (40×40 ×3 cm³)containing 1.6 l deionized water. These seeds were then cultured at 25°C. for 2 days in the dark under aeration with an air pump.

(2) Purification of the present compounds

1 Each seed immersion solution obtained in (1) above was filteredthrough Toyo No. filter paper, and each filtrate was concentrated at 35°C. under reduced pressure. Then, said concentrate was partitioned intoacetone soluble and insoluble phases.

Amaranthus caudatus L. seeds were gently placed on a filter paperimmersed with 0.8 ml of a sample solution (from an acetone soluble orinsoluble fraction) in a Petri dish of 3 cm diameter and were thenallowed to stand at 25° C. for 5 days in the dark, and the lengths ofgerminated hypocotyls and roots were determined for the evaluation ofwhether or not any biological activity on plant growth is present in theacetone soluble and insoluble phases.

As a result, the activity showing the growth promotion of hypocotyls andthe growth inhibition of roots (of Amaranthus caudatus L.) was confirmedto be present in the acetone insoluble fraction.

Subsequently, the acetone insoluble fraction was dissolved in 10 mlwater and then separated by molecular exclusion chromatography (Mol cut,Millipore Corp.) into three fractions, i.e. M_(r) above 100,000,5,000-100,000, and below 5,000. The biological activity was found to bepresent in the fraction of M_(r) below 5,000. This fraction wasconcentrated at 35° C. under reduced pressure.

2 Each concentrate (about 150 mg) obtained in 1 above was dissolved inwater and purified by HPLC (Waters, μ Bondasphere 5μ C₁₈ -100Å; column19 mm×15 cm; eluent 100% H₂ O, flow rate 5 ml/min; 214 nm detector). Theabove biological activity was found in fractions of retention time of5-8 minutes. The retention time agreed with the retention time (in HPLCunder the same conditions) of the compound obtained in Example 1 fromthe cress seedlings.

Said HPLC fractions were combined and concentrated at 35° C. underreduced pressure and further purified by HPLC (YMC Packed Column AQ-324S-5 120A ODS; SiC Co. Ltd.; eluent 100% H₂ O. flow rate 1 ml/min; 214 nmdetector). The above biological activity was found in fractions ofretention time of 17.0-17.8 minutes. These fractions were combined andconcentrated at 35° C. under reduced pressure, whereby a small amount ofamorphous powder (several mg) originating in each plant was obtained.

(3) Determination of biological activities of the present compounds

1 Out of the above purified compounds, the compound derived fromsunflower seeds was mixed with 47 g vermiculite, and this vermiculitewas placed in a vessel of 9 cm diameter, and Amaranthus caudatus L.,lettuce, Persian speedwell, and timothy seeds were scattered in thisvessel and grown at 25° C. over 3 weeks in the sunlight for 16 hours perday.

The result indicated that the hypocotyl growth of every plant waspromoted about 2 times as high as the corresponding control at each leftin (A) to (D), as can be seen from the photograph of seedlings in FIG.2. In addition, it was made evident that at a low concentration of 100ppm, the growth of roots excluding those of the simple leaf planttimothy was also promoted about 2 times as high as the control.

2 Each of the above compounds (in an amount of the compound derived from10 seeds) originating in cress seeds and 19 kinds of plants (sunfloweretc.) seeds was dissolved in 1 ml distilled water, and the samplesolution was then put in a vessel of 3.3 cm diameter without a filterpaper, and Amaranthus caudatus L. seeds were scattered thereon and grownat 25° C. for 5 days in the dark.

As a result, the hypocotyl growth of a number of plants, similar to 1above, was promoted even in this experimental system, as shown in FIG.3. The growth promotion activity was relatively low in the cases oflettuce, Amaranthus caudatus L., tomato, and Persian speedwell, but thesame growth promotion activity was exhibited by increasing 10 times ashigh concentration as the original, so that the growth promotionactivity is considered to be a common phenomenon among said 19 kinds ofplants.

(Example 3) Promotion activity of the hypocotyl elongation of Amaranthuscaudatus L. seedlings in a secretion solution from germinated seeds

10 seeds each of 44 kinds of plants, slender amaranth etc., weresterilized for 30 minutes in 1% aqueous sodium hypochlorite solution,followed by washing with tap water for 30 minutes and then withdistilled water. The moist seeds were placed in a vessel (3.3 cmdiameter) containing 1 ml water and were then cultured at 25° C. in thedark. In the case of large seeds such as corn, pea, and oat seeds, avessel of 6 cm diameter was employed and 3 ml distilled water was added.After 2 days of culture, the secretion solution was transferred to avessel with a filter paper (Toyo filter paper No. 1), and 10 seeds ofAmaranthus caudatus L. were scattered. After cultured at 25° C. for 5days in the dark, the length of Amaranthus caudatus L. hypocotylsgerminated was determined. In case plant seeds indicated little or nohypocotyl growth promotion in the above test, a few secretion solutionswere prepared from a greater number of seeds, e.g. 50. 100, or 150seeds, and were used in an Amaranthus caudatus L. hypocotyl elongationtest. As the control, distilled water was used in place of the secretionsolution.

The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                                    Amaranthus caudatus L. hypocotyl                                              elongation (mm ± S.E.)                                         Plants        10 seeds  50 seeds  100 seeds                                   ______________________________________                                        slender amaranth                                                                             7.8 ± 1.5                                                                            8.3 ± 1.0                                                                           10.9 ± 0.8                               asparagus     11.1 ± 1.4       15.3 ± 2.0                               barnyard grass                                                                              15.9 ± 3.1                                                   rice          15.0 ± 2.9                                                   green foxtail 15.0 ± 1.9                                                   pea           19.5 ± 2.8                                                   timothy grass  7.9 ± 1.6       11.8 ± 1.4                               Persian speedwell                                                                           11.5 ± 2.0                                                                           13.3 ± 1.7                                         okra          21.8 ± 5.4                                                   wild oat      20.7 ± 3.0                                                   goose grass    7.9 ± 1.7                                                                           11.6 ± 1.1                                         turnip         8.2 ± 1.6       12.9 ± 2.7                               pumpkin       17.2 ± 4.0                                                   cauliflower    9.2 ± 2.4       12.2 ± 1.2                               cabagge        5.6 ± 1.0                                                                           10.8 ± 1.6                                         cress         19.4 ± 1.5                                                   cockspur grass                                                                               7.7 ± 1.9                                                                           12.5 ± 1.5                                         rice flat-sedge                                                                              8.7 ± 1.5                                                                           10.7 ± 1.4                                         burdock       28.5 ± 4.8                                                   "shikokubie"  15.6 ± 2.4                                                   perilla       12.4 ± 2.5       18.0 ± 2.2                               crown daisy   17.8 ± 3.6                                                   common purslane                                                                              9.7 ± 2.0                                                                           10.5 ± 1.3                                         celery         8.9 ± 2.0       20.0 ± 3.2                               buckwheat     21.7 ± 3.2                                                   radish        21.6 ± 2.8                                                   "ta-inubie"   17.2 ± 2.1                                                   small-flower umbrella-                                                                       9.2 ± 2.3                                                                           10.2 ± 1.0                                         plant                                                                         corn          19.0 ± 2.5                                                   tomato        11.0 ± 2.9                                                                           16.9 ± 3.0                                         eggplant      16.2 ± 2.8                                                   leek          19.8 ± 4.3                                                   carrot        17.7 ± 3.6                                                   chinese cabbage                                                                              8.1 ± 2.7       12.4 ± 2.0                               parsley       27.3 ± 4.9                                                   sunflower     20.9 ± 4.9                                                   amaranth       7.8 ± 1.6                                                                           12.8 ± 1.9                                         broccoli      13.4 ± 2.7                                                   spinach       34.8 ± 5.4                                                   hardstem bulrush                                                                             9.0 ± 2.1                                                                            9.6 ± 1.0                                                                           *14.5 ± 2.8                              matricaria     6.8 ± 1.7                                                                           10.7 ± 1.5                                         trefoil       19.7 ± 4.3                                                   cabgrass      12.7 ± 2.1                                                   lettuce        7.8 ± 1.5                                                                           10.0 ± 1.0                                         Control        8.3 ± 1.5                                                                            7.8 ± 1.0                                                                            7.6 ± 1.2                               ______________________________________                                         *150 seeds were used.                                                    

The secretion solutions from spinach, burdock, and parsley exhibited aparticularly strong promotion activity of hypocotyl elongation. Thesecretion solutions from 13 kinds of plants, i.e. buckwheat, radish,sunflower, oat, leek, trefoil, pea, cress, corn, okra, crown daisy,carrot. "ta-inubie" exhibited a relatively strong activity. Thesecretions solution from 20 kinds of plants, i.e. slender amaranth,asparagus, timothy grass, etc., exhibited a weak activity.

From the above results, 44 kinds of plants set forth in Table 1 provedto secret the present compound (1) having a hypocotyl elongationpromotion activity, and it is understood that the present compound (1)can be obtained by extraction of their seeds or from their secretionsolution.

(Example 4) Hydroponics in the presence of the present compound

A. Preparation of a sunflower secretion solution

A secretion solution was prepared from 2000 sunflower seeds using 2 lwater in the same manner as in Example 2. The secretion solution wasconcentrated under reduced pressure, whereby four concentrated extractedsolution of a predetermined concentration expressed in terms of thenumber of sunflower seeds (50 to 200 seeds) were prepared, and the finalvolume of each aqueous solution was adjusted to 20 ml for use in ahydroponics experiment.

B. Method for hydroponics

Tomato, lettuce, pea, and cabbage were selected as test plants, andtheir seeds were seeded and then incubated at 25° C. for 2 days (3 daysfor tomato) in the cycle of 12 hours in the dark and 12 hours in thebright. Then, the seedlings were transplanted to a hydroponics chambermade of glass (28 mm diameter, 40 mm height, and 20 ml volume) andsubjected to hydroponics for 7 days at 25° C. in the cycle of 12 hoursin the bright and 12 hours in the dark. The seedlings were then examinedfor lower and upper hypocotyl length, root length, leaf size, andweight.

(1) Lettuce hydroponics

According to the same method as described above, a hydroponicsexperiment was made using four secretion solutions containing thepresent compound (1) (referred to as "lepidimoide", hereinafter) at apredetermined concentration expressed in terms of the number ofsunflower seeds (i.e., 50, 100, 150, and 200 seeds) and alepidimoide-free control. 7 days thereafter, the weight and length ofhypocotyls and roots were determined for the evaluation of the effect onplant growth.

The results are set forth in Tables 2 and 3 and FIG. 4.

                  TABLE 2                                                         ______________________________________                                        Measurement results of hypocotyl and root lengths                             Hypocotyl length                                                                         Hypocotyl                                                                             Root length                                                Length of    elongation                                                                              Length of                                              part of      ratio (based                                                                            part of   Root elongation                              plant over   on the    plant under                                                                             ratio (based on                              the ground   control as                                                                              the ground                                                                              the control as                               (mm)         100%)     (mm)      100%)                                        ______________________________________                                        Control                                                                               8.1      100       27.4    100                                        50 seeds                                                                             10.0      128       65.4    239                                        100 seeds                                                                            10.6      131       69.1    252                                        150 seeds                                                                             9.3      115       73.6    269                                        200 seeds                                                                            11.9      147       67.5    248                                        129% on average    251% on avarage                                            ______________________________________                                         *Length is the average of 8 seedings.                                    

                  TABLE 3                                                         ______________________________________                                        Measurement results of weight increase                                        (average weight of 8 seedlings) (unit: mg)                                    Weight of               Weight of                                             part of                 part of                                               plant over    Increase  plant under                                                                              Increase                                   the ground    ratio     the ground ratio                                      ______________________________________                                        Control                                                                               61.1      100%      15.8     100%                                     50 seeds                                                                             142.9      234%      49.9     315%                                     100 seeds                                                                            156.0      255%      51.7     327%                                     150 seeds                                                                            140.4      230%      55.5     351%                                     200 seeds                                                                            175.7      288%      72.8     460%                                     252% increase on average                                                                          363% increase on average                                  ______________________________________                                    

FIG. 4 shows the results of 3 days hydroponics of seedlings in wateronly (control) and those in water containing lepidimoide in an amountderived from 50, 100, 150, and 200 sunflower seeds (from left to right).From the results, a significant growth promotion effect can be seen onthe hypocotyls and roots after the third day of hydroponics.

It was confirmed by Table 2 that after the hydroponics of lettuce,lepidimoide elicited 29% augmentation on the average and 47%augmentation at the maximum in the elongation of hypocotyls and 151%augmentation on the average in the elongation of roots. The greater thenumber of seeds used for extraction (i.e. the higher the concentrationof lepidimoide), the higher the elongation rate was observed. As can beseen from Table 3, lepidimoide elicited 252% augmentation on the average(approx. 2.5 times) and 288% augmentation at the maximum (approx. 2.9times) in the weight of hypocotyls. Furthermore, there were observed363% augmentation on the average (approx. 3.6 times) and 460%augmentation (approx. 4.6 times) at the maximum in the weight of roots,so that lepidimoide proved to exhibit a significant promotion effect onan increase in weight. It became evident within the concentration rangeof lepidimoide tested that the higher the concentration of lepidimoideadded, the higher the effect on an increase in the weight of bothhypocotyls and roots.

(2) Pea hydroponics

According to the method as described above, a hydroponics test wasconducted in the same manner as for lettuce, using 4 secretion solutionscontaining lepidimoide at a predetermined concentration, and the growthpromotion effect was examined after 7 days culture.

The results are shown in Tables 4 and 5 and FIG. 5.

                  TABLE 4                                                         ______________________________________                                        Measurement results of hypocotyl and root weights                             (average of 4 seedlings) (after 7 days)                                                    Weight-                                                                       increase                                                         Weight of    ratio in  Weight of Weight-increase                              part of      part of   part of   ratio in part                                plant over   plant over                                                                              plant under                                                                             of plant under                               the ground   the ground                                                                              the ground                                                                              the ground                                   ______________________________________                                        Control                                                                              602.3     100%       679.7  100%                                       50 seeds                                                                             724.9     120%      1067.4  157%                                       100 seeds                                                                            802.7     133%      1175.9  173%                                       150 seeds                                                                            863.7     143%      1078.6  159%                                       200 seeds                                                                            1033.9    172%      1208.3  178%                                       142% increase on   167% increase on average                                   average                                                                       ______________________________________                                    

                  TABLE 5                                                         ______________________________________                                        Measurement results of hypocotyl and root lengths                             (average weight of 4 seedlings) (after 7 days)                                Length of               Length of                                             part of                 part of                                               plant over    Elongation                                                                              plant under                                                                             Elongation                                  the ground    ratio     the ground                                                                              ratio                                       ______________________________________                                        Control                                                                              41.3       100%      51.5    100%                                      50 seeds                                                                             47.3       115%      55.8    108%                                      100 seeds                                                                            59.5       144%      65.8    128%                                      150 seeds                                                                            60.0       145%      72.3    140%                                      200 seeds                                                                            74.5       180%      85.3    146%                                      146% increase on average                                                                          131% increase on average                                  ______________________________________                                    

FIG. 5 is a photograph of seedlings after 3 days hydroponics. It isunderstood that the growth of 3 seedlings (from the right) cultured inthe presence of lepidimoide at the same concentrations as in FIG. 4 ispromoted as compared with the control (left).

When cultured in water containing lepidimoide (i.e., a sunflowersecretion solution), the elongation of pea hypocotyls was approx. 1.5times on the average and 1.8 times at the maximum as high as that of thecontrol, and the elongation of pea roots was approx. 1.3 times on theaverage and 1.5 times at the maximum as high as that of the control, ascan been seen from Table 4. In addition, the weight increase of peahypocotyls was approx. 1.4 times on the average and 1.7 times at themaximum as much as that of the control, and the weight increase of pearoots was approx. 1.7 times on the average and 1.8 times at the maximumas much as that of the control. Particularly within the concentrationrange of lepidimoide tested, it was made evident that the higher theconcentration of lepidimoide added, the higher the effect on theelongation and weight increase of both hypocotyls and roots. Asignificant growth promotion effect was observed within the lepidimoideconcentration range in this example.

(3) Tomato hydroponics

According to the method as described above, a hydroponics experiment wascarried out using water containing lepidimoide (i.e., the sunflowersecretion solution) at 3 predetermined concentrations for theexamination of growth promotion effect (after 7 days of culture).

The results are set forth in Tables 6 and 7 and FIG. 6.

                  TABLE 6                                                         ______________________________________                                        Measurement results of hypocotyl and root lengths                             (average of 8 seedlings)                                                      Length of               Length of                                             part of                 part of                                               plant over              plant under                                           the ground    Elongation                                                                              the ground                                                                              Elongation                                  (mm)          ratio (%) (mm)      ratio (%)                                   ______________________________________                                        Control  7.9      100%      27.4    100%                                      50 seeds                                                                              17.2      218%      37.9    138%                                      100 seeds                                                                             15.4      195%      17.1     62%                                      200 seeds                                                                             12.7      163%      11.9     43%                                      ______________________________________                                    

                  TABLE 7                                                         ______________________________________                                        Measurement results of hypocotyl and root weights                             Weight of               Weight of                                             part of                 part of                                               plant over              plant under                                           the ground    Increase  the ground                                                                              Increase                                    (mg)          ratio (%) (mg)      ratio (%)                                   ______________________________________                                        Control  6.8      100%      26.4    100%                                      50 seeds                                                                              207.3     305%      88      333%                                      100 seeds                                                                             142.4     209%      26.6    101%                                      200 seeds                                                                              65.9      97%      28.4    108%                                      ______________________________________                                    

As compared with the control (left), the growth of the seedlingsincubated in the presence of lepidimoide at the same concentrations asin FIGS. 4 and 5 proved to be significantly promoted even after 3 daysof culture, as shown in FIG. 6. FIGS. 6 and 7 indicate that the presenceof lepidimoide was greatly affective for increases in the length andweight of tomato hypocotyls and roots. The hypocotyl and root lengthswere approx. 2.2- and 1.4 times respectively at the maximum as long asthose of the control. In addition, lepidimoide proved to exhibit asignificant effect on increases in hypocotyl and root weights. That is,the hypocotyl and root weights were approx. 1.3- and 3.1 timesrespectively at the maximum as much as those of the control.

On the other hand, it was found that lepidimoide, at a higherconcentration, inhibits root elongation but does not inhibit root weightincrease.

(4) Cabbage hydroponics

According to the method as described above, a hydroponics experiment wasconducted using water containing lepidimoide (i.e. the sunflowersecretion solution) at 3 predetermined concentrations for theexamination of growth promotion effect (after 7 days of culture).

The results are set forth in Tables 8 and 9.

                  TABLE 8                                                         ______________________________________                                        Measurement result of hypocotyl and root lengths                              (average of 8 seedlings)                                                      Length of               Length of                                             part of                 part of                                               plant over              plant under                                           the ground    Elongation                                                                              the ground                                                                              Elongation                                  (mm)          ratio (%) (mm)      ratio (%)                                   ______________________________________                                        Control 14.0      100%      15.1    100%                                      50 seeds                                                                              30.8      220%      28.5    189%                                      100 seeds                                                                             25.6      183%      16.3    108%                                      200 seeds                                                                             24.1      172%      14.3     75%                                      ______________________________________                                    

                  TABLE 9                                                         ______________________________________                                        Measurement results of hypocotyl and root weights                             (average of 8 seedlings)                                                      Weight of               Weight of                                             part of                 part of                                               plant over              plant under                                           the ground    Increase  the ground                                                                              Increase                                    (mg)          ratio (%) (mg)      ratio (%)                                   ______________________________________                                        Control 50.0      100%      367.6   100%                                      50 seeds                                                                              102.7     206%      444.2   122%                                      100 seeds                                                                             55.8      112%      288.9    79%                                      200 seeds                                                                             --        --        232.7    63%                                      ______________________________________                                    

From Tables 8 and 9, it was confirmed that as a result of the growthpromotion effect of lepidimoide, cabbage hypocotyl and root elongationswere approx. 2.2- and 1.9 times respectively at the maximum as high asthose of the control, and the cabbage hypocotyl and root weights wereapprox. 2- and 1.2 times respectively at the maximum as those of thecontrol.

In summary, it was confirmed that a method for hydroponics usinglepidimoide (or a lepidimoide-containing secretion solution fromsunflower seeds) is considerably effective for the elongation and weightincrease of lettuce, pea, tomato, and cabbage hypocotyls and roots.

It was proved by the above data that lepidimoide applied to hydroponicsbrings about the significant growth promotion of plants, resulting in areduction in a period of time required for culture as well as animprovement in growth rate.

Hence, plant growth can be greatly improved according to hydroponicsusing lepidimoide.

(Example 5) Growth inhibition of black matpe roots

Sunflower seeds were used for the preparation of alepidimoide-containing stock solution. According to the method asdescribed in Example 2, a stock solution was prepared by extraction of2000 sunflower seeds with 2 1 water, and it was used as a standard stocksolution to be concentrated or diluted as necessary.

One type of moyashi seeds, black seeds (black matpe), was used as asubject plant in an experiment of root growth inhibition usinglepidimoide at a concentration expressed in terms of the number ofsunflower seeds (0 [control], 150, 500, and 1500 seeds).

500 ml black matpe seeds were immersed at 40° C. for 4 hours in 3 l ofan aqueous 20 ppm sodium hypochlorite solution containing lepidimoide.The seeds were then transferred to a plastic vessel of specific volumeand grown at 25° C. for 3 days in the dark. During culture, water at 15°C. was spread intermittently at an interval of 8 hours.

FIG. 7 shows the results of the growth inhibition experiment in whichlepidimoide was used at a concentration of 0 (control), 150, 500, and1500 sunflower seeds (from left to right). As is evident from thisphotograph, the growth inhibition of roots is observed to increase fromleft to right. In particular, the growth inhibition of roots issignificant in the case of 1500 seeds at the right, and their rootlength was found to be approx. 20% shorter than that of the control.

The growth inhibition effect of lepidimoide on moyashi roots wasconfirmed from the above results.

(Example 6) Growth inhibition effect of lepidimoide on black matpe andgreen gram roots under different treatment conditions

Since the growth inhibition effect of lepidimoide on roots was confirmedin Example 5, optimum conditions etc. for lepidimoide treatment weredetermined under similar experimental conditions, and black matpe andgreen gram roots were examined for growth inhibitory effect. Culture wascontinued for 5 days, and the concentration of lepidimoide was approx.twice as high as that in Example 5. The experiment comprises 5treatments: Treatment 1 involves immersion in a lepidimoide solutionevery day; treatment 2 immersion on the first day only; treatment 3immersion on the first and second days; treatment 4 immersion on thefirst and third days; and treatment 5 immersion on the first and fourthdays. On the fifth day, 30 moyashi were arbitrarily picked up from eachof the treatment groups, and their hypocotyl and root lengths on theaverage were determined and compared. The results are set forth inTables 10 and 11.

                  TABLE 10                                                        ______________________________________                                        Growth inhibition effect on black matpe roots                                              Treat-  Treat-  Treat-                                                                              Treat-                                                                              Treat-                               Control      ment 1  ment 2  ment 3                                                                              ment 4                                                                              ment 5                               ______________________________________                                        Hypocotyls                                                                            51.4     51.5    49.0  47.3  47.0  50.0                               Roots   20.6     17.9    14.9  23.5  20.7  20.6                               ______________________________________                                    

                  TABLE 11                                                        ______________________________________                                        Growth inhibition effect on green gram roots                                               Treat-  Treat-  Treat-                                                                              Treat-                                                                              Treat-                               Control      ment 1  ment 2  ment 3                                                                              ment 4                                                                              ment 5                               ______________________________________                                        Hypocotyls                                                                            43.9     46.8    43.1  45.2  48.2  49.0                               Roots   30.6     17.5    16.8  21.7  19.2  16.3                               ______________________________________                                    

From the experimental results in Table 10, it was reconfirmed thatlepidimoide treatment is effective for the growth inhibition of blackmatpe roots. Lepidimoide treatment every day or on the first day waseffective, as can be seen from Table 10. Treatment 2 is a simple andeconomical method since immersion suffices only 1 time and 28%inhibitory effect at the maximum on root growth was achieved.

From the experimental results in Table 11, treatments 1 to 5 proved tobe effective for the growth inhibition of green gram roots, and thegrowth inhibition of roots by lepidimoide proved to be effectiveparticularly for green gram. In addition, 47% inhibition effect at themaximum was observed. 45% inhibition effect was observed even intreatment 2 where immersion was carried out only 1 time, so thattreatment 2 is understood to be a most reasonable and economicaltreatment.

In the case of green gram, the effect on hypocotyl elongation (i.e. thephysiological effect of lepidimoide as described hereinbefore) was alsoobserved to some extent.

From the above experiments using black matpe and green gram, it wasfound that lepidimoide can be used to inhibit the growth of moyashiroots, and it is effective and reasonable to treat moyashi withlepidimoide at the first stage of growth. In this case, treatment 2,where immersion suffices only one time, proved to be economical andsuitable for application to the actual production process.

(Example 7) Growth promotion of moyashi by lepidimoide

As described above, lepidimoide proved to have an effect on hypocotylelongation, and as shown in data of Example 6, it was expected thatlepidimoide is effective for the growth promotion of green gram. Hence,the growth promotion effect of lepidimoide on green gram was evaluatedunder the same conditions as in Example 5. The concentration oflepidimoide was 1000 sunflower seeds/l, and green gram was cultured for7 days.

The results are set forth in FIG. 8. The left seedlings were treatedwith lepidimoide, and the right seedlings (control) were not treated. Itis confirmed by this photograph that the seedlings treated withlepidimoide (left) are approx. twice as large as the untreated seedlings(right). This results indicate the significant growth promotion effectof lepidimoide.

From the above experiments, the significant growth promotion effectbrought about by treatment with lepidimoide is expected to significantlyimprove moyashi production rate and productivity, resulting in areduction in production costs, and lepidimoide treatment is extremelyuseful for the large scale production of moyashi.

(Synthesis Example 1) Benzylation of α-L-rhamnose ##STR10##

100 ml benzene was added to 4 g of α-L-rhamnose monohydrate in 20 mlbenzyl alcohol, and the mixture was heated for 2 hours under reflux inthe presence of a catalytic amount of sulfuric acid (20 drops from aPasteur pipette), and the resulting water was azeotropically removed.The reaction solution was concentrated under reduced pressure (approx.40° C.) and purified by column chromatography on silica gel (50 g silicagel, chloroform:methanol=20:1), whereby benzyl glycoside, 3.7 g, wasobtained (yield: 93%).

(Synthesis Example 2) Synthesis of compound (2) ##STR11##

5.5 g of the benzyl glycoside obtained in Synthesis Example 1 and 10 mlof 2,2-dimethoxypropane were added to 90 ml acetone and allowed to reactfor 1.5 hours at room temperature in the presence of 2.0 g ofp-toluenesulfonic acid as a catalyst. The reaction solution wasconcentrated under reduced pressure, followed by addition of 100 mlethyl acetate. Subsequently, the sample was washed with an aqueous,saturated sodium hydrogen carbonate solution (50 ml), then water (50ml×2), and an aqueous, saturated sodium chloride solution (50 ml), andthe product was then dried over sodium sulfate anhydride, concentrated,and purified by column chromatography on silica gel (50 g silica gel,chloroform:methanol: 50:1), whereby acetonide (2), 4.9 g, was obtained(yield: 89%).

(Synthesis Example 3) Synthesis of compound (3) ##STR12##

15 ml N,N-dimethylformamide (DMF) containing 4 g of the alcoholderivative (2) obtained in Synthesis Example 2 was added to 10 mlsuspension of 0.82 g sodium hydride in DMF, and the mixture was stirredat 0° C. for 5 minutes and at room temperature for 45 minutes. Thereaction solution was cooled to 0° C. again, followed by addition of 3.4benzyl bromide. The mixture was stirred at 0° C. for 40 minutes and atroom temperature for 39 hours. To the reaction solution (0° C.) wereadded 0.5 ml methanol and then 50 ml water, and the product wasextracted with ethyl acetate (200 ml). The organic layer was washed withan aqueous, saturated sodium chloride solution (100 ml×2) then driedover sodium sulfate anhydride, and concentrated. The concentrate waspurified by chromatography on silica gel (100 g silica gel, hexane:ethylacetate=5:1), whereby benzyl derivative (3), 3.4 g, was obtained (yield:84.2%).

(Synthesis Example 4) Synthesis of compound (4) ##STR13##

1,4-Dioxane, acetic acid, and water, 30 ml each, were added to 4 g ofthe benzyl derivative (3) obtained in Synthesis Example (3), and themixture was allowed to react at 70°-75° C. for 3.5 hours. The reactionsolution was poured into 300 ml ethyl acetate, and the organic layer waswashed with water (150 ml), an aqueous, saturated sodium hydrogencarbonate solution (100 ml), and an aqueous, saturated sodium chloridesolution (200 ml×2), then dried over sodium sulfate anhydride, andconcentrated. For purification, the product was fractionated by columnchromatography on silica gel (60 g silica gel, hexane:ethyl acetate=5:1to 1:1), whereby compound (4), 2.8 g, was obtained (yield: 70%).

(Synthesis Example 5) Synthesis of compounds (5) and (6) ##STR14##

A mixture of 2.24 g of compound (4) obtained in Synthesis Example 4 and1.82 g dibutyltin oxide (Bu₂ SnO) in 30 ml abs. benzene was heated over3 hours under reflux, and the resulting water was azeotropicallyremoved. The reaction solution was concentrated and then dried for 1hour with a vacuum pump, so that compound (5) was obtained. 1.5 g cesiumfluoride (CsF) was added to compound (5) in situ, and the sample wasthen dried for another 1 hour with a vacuum pump, followed by additionof 30 ml DMF and then 3 ml benzyl bromide. The mixture was allowed toreact for 1.5 hours, and then the reaction solution was poured into 100ml ethyl acetate. The organic layer was washed with water (100 ml×2) andthen with an aqueous, saturated sodium chloride solution (100 ml), andthe solution was dried over sodium sulfate anhydride and concentrated.For purification, the product was fractionated by column chromatographyon silica gel (90 g silica gel, hexane: ethyl acetate: 5:1 to 2:1),whereby compound (6), 2 g, was obtained (yield in the 2-step reaction:88%).

(Example 8) Synthesis of compound (7) ##STR15##

6 g molecular sieve A was added to a mixture of 600 mg compound (A) and1114 mg of compound (6) obtained in Synthesis Example 5, and the mixturewas dried under reduced pressure with a vacuum pump. 3 hours thereafter,20 ml abs. dichloromethane was added thereto, followed by 1 hourstirring. Subsequently, 2.5 g methylsulphenylbromide (MSB) in 5 ml1,2-dichloroethane was added at 0° C. in the dark to the mixture, whichin turn was stirred for 1 hour. The reaction temperature was lowered to0° C., followed by addition of 4 ml triethylamine and then 50 ml ethylacetate. The reaction solution was filtered, dried over sodium sulfateanhydride, and concentrated. The product was purified by columnchromatography on silica gel (160 g silica gel, hexane:ethyl acetate:7:1 to 7:3), whereby compound (7), 1400 mg, was obtained (yield: 37.5%).

IR spectrum: see FIG. 9.

¹ H-NMR spectrum (CDCl₃): see FIG. 10.

¹³ C-NMR spectrum (CDCl₃): see FIG. 11.

(Example 9) Synthesis of compound (8) ##STR16##

300 mg of compound (7) obtained in Example 8 was dissolved in 6 mlmethanol, and 100 mg potassium carbonate was added thereto, and themixture was stirred at room temperature for 1 hour and 20 minutes. Thereaction solution was poured into ethyl acetate, and the organic layerwas washed with an aqueous, saturated sodium chloride solution (40ml×3), dried over sodium sulfate anhydride, and concentrated. Forpurification, the product was separated by thin layer chromatography,whereby compound (8), 285mg, was obtained (quantitatively).

(Example 10) Synthesis of compound (8a) ##STR17##

10 mg of compound (8) obtained in Example 9 was dissolved in 3 mlmethanol. The air in the reaction vessel was degassed and replaced byargon, and 10% palladium-carbon was added at 0° C. thereto, and then theargon in the vessel was replaced by hydrogen at room temperature. Themixture was then stirred for 15 hours at atmospheric pressure. Thehydrogen in the vessel was replaced by argon After addition of Celite,the reaction mixture was filtered. The filtrate was concentrated andthen purified by preparative silica gel chromatography (chloroform:methanol=2:1), whereby compound (8a), 9 mg, was obtained (yield: 90%).

¹ H-NMR spectrum (CD₃ OD): see FIG. 12.

(Example 11) Synthesis of compound (9) ##STR18##

240 mg of compound obtained in Example 9 was dissolved in a combinedsolvent of 2 ml DMSO and 0.8 ml triethylamine, and 400 mg SO₃ -pryridinewas added little by tittle. The reaction solution was stirred for 4hours at room temperature and was then poured into water. Afterextracted with 30 ml ethyl acetate, the organic layer was washed with anaqueous, saturated sodium chloride solution (15 ml×2), dried over sodiumsulfate anhydride, and concentrated.

230 mg of this concentrate, 1 ml of 2-methyl-2-butene, and 50 mg ofsodium dihydrogen phosphate were dissolved in a combined solvent of 2 mlwater and 3 ml of t-butyl alcohol. 170 mg of sodium chlorite (approx.85% NaClO₂) was added little by little, and then the mixture was thenstirred for 2 hours. The reaction solution was cooled to 0° C. andpoured into 30 ml ethyl acetate. Subsequently, the solution was madeacidic with 1N hydrochloric acid and then washed with an aqueous,saturated sodium chloride solution (20 ml×3), dried over sodium sulfateanhydride, and concentrated. The residue was purified by thin layerchromatography, whereby carboxylic acid (9), 190 mg, was obtained (yieldin the two-step reaction: 79%).

(Example 12) Synthesis of compound (9a) ##STR19##

20 mg of compound (9) obtained in Example 11 was dissolved in 5 mlmethanol. The air in the reaction vessel was degassed and replaced byargon, and 10% palladium-carbon was added at 0° C. thereto, and then theargon in the vessel was replaced by hydrogen at room temperature. Themixture was then stirred for 15 hours at atmospheric pressure.

The hydrogen in the vessel was replaced by argon. After addition ofCelite, the reaction mixture was filtered. The filtrate was concentratedand then purified by preparative silica gel chromatography(chloroform:methanol: 1:1), whereby compound (9a), 18 mg, was obtained(yield: 90%).

¹ H-NMR spectrum (CD₃ OD): see FIG. 13.

(Example 13) Synthesis of compound (10) ##STR20##

200 mg of compound (9) obtained in Example 11 was dissolved in a mixtureof 5 ml benzene and 1 ml methanol, followed by addition of an excessamount of trimethylsilyldiazomethane (in 10%; benzene). 5 minutesthereafter, the solvent was evaporated, whereby compound (10), 190 mg,was obtained (quantitatively).

(Example 14) Synthesis of compound (10a) ##STR21##

174 mg of compound (10) obtained in Example 13 was dissolved in acombined solvent of 10 ml methanol and 10 ml ethyl acetate. The air inthe reaction vessel was degassed and replaced by argon, and 10%palladium-carbon was added at 0° C. thereto, followed by replacement atroom temperature of the argon by hydrogen. The mixture was then stirredfor 15 hours at atmospheric pressure, and the hydrogen in the vessel wasreplaced by argon. After addition of Celite, the reaction mixture wasfiltered, and the filtrate was concentrated and purified by preparativechromatography on silica gel (chloroform:methanol=2:1), whereby alcoholderivative (10a), 170 mg, was obtained (yield: 98%).

IR spectrum: see FIG. 14.

¹ H-NMR spectrum (CD₃ OD): see FIG. 15.

¹³ C-NMR spectrum (CD₃ OD): see FIG. 16.

(Example 15) Synthesis of compound (11) ##STR22##

65 mg of the hexalcohol derivative (10a) obtained in Example 14 wasdissolved in 0.8 ml pyridine, and 0.5 ml acetic anhydride was addedthereto. Then, the mixture was stirred for 11 hours at room temperature.After addition of toluene, the solvent was azeotropically removed, andthe residue was purified by preparative chromatography on silica gel(chloroform:acetone: 5:1), whereby compound (11), 62 mg, was obtained(quantitatively).

(Example 16) Synthesis of compounds (12) and (12') ##STR23##

15 mg of 1,8-diazabicyclo[5.4.0]-7-undecene (DBU) was added to 0.5 mlpyridine containing 1.3 mg of compound (11) obtained in Example (15),and the mixture was stirred under cooling on ice for 1 hour and at roomtemperature for additional 14 hours. The reaction solution was pouredinto 30 ml ethyl acetate and was then washed with 5 ml of 1Nhydrochloric acid and then with an aqueous, saturated sodium chloridesolution (15 ml×3). The sample was dried over sodium sulfate anhydrideand concentrated. The residue was purified on thin layer chromatography,whereby 0.7 mg of compound (12) and 0.6 mg of compound (12') wereobtained (yield (12): 54%, (12'): 43%). Compound (12):

State: Colorless oil residue

Mass spectrum: m/z 546.1564 (M⁺)

IR spectrum (film): 1740 cm⁻¹ (see FIG. 17)

¹ H-NMR spectrum: δ (CDCl₃) (see FIG. 18)

6.14 (1H, d, J=3.3 Hz), 6.04 (1H, d, J=2.0 Hz) 5.59 (1H, dd, J=7.5, 3.3Hz), 5.31 (1H, d, J=2.4 Hz) 5.18 (1H, dd, J=7.5, 2.4 Hz), 5.16 (1H, dd,J=9.8, 3.4 Hz), 5.06 (1H, dd, J=9.8, 9.3 Hz), 4.27 (1H, dd, J=3.4, 2.0Hz), 3.88 (1H, dq, J=9.3, 6.0 Hz), 3.80 (3H, s), 2.14 (6H, s), 2.10 (3H,s) 2.05 (3H, s), 2.00 (3H, s), 1.22 (3H, d, J=6.0 Hz)

¹³ C-NMR spectrum: (CDCl₃) (see FIG. 19) 170.4(s), 170.1(s), 169,9(s),169.5(s), 168.8(s), 161.6(s), 141.6(s), 108. 6(d), 95.6(d), 90.2(d),73.3(d), 70.4(d), 69.4(d), 69.0(d), 68.3(d), 66.2(d), 52.5(q), 20.94(q),20.90(q), 20.7(q), 20.6(q), 20.5(q), 17.5(q) Compound (12'):

State: Colorless oil residue

Mass spectrum: m/z 546.1609 (M⁺)

IR spectrum (film): 1740 cm⁻¹ (see FIG. 20)

¹ H-NMR spectrum: δ (CDCl₃) (see FIG. 21)

6.15 (1H, d, J=2.9Hz), 5.72 (1H, s), 5.71 (1H, d, J=2.9 Hz), 5.64 (1H,dd, J=7.8, 2.9 Hz), 5.10 (1H, dd, J=7.8, 2.9 Hz), 5.02 (1H, dd, J=9.8,9.8 Hz), 4.91 (1H, dd, J=9.8, 3.2 Hz), 4.43 (1H, d, J=3.2 Hz), 3.59 (1H,dq, J=9.8, 6.1 Hz), 3.80 (3H, s), 2.13 (3H, s), 2.12 (3H, s), 2.10 (3H,s), 2.04 (3H, s), 1.95 (3H, s), 1.25 (3H, d, J=6.1 Hz)

(Example 17) Synthesis of compound (1) ##STR24##

3.1 mg of compound (12') obtained in Example 16 was dissolved in acombined solvent of 1 ml water and 1 ml methanol, followed by additionof 0.034 ml of 50 % methanol (aq.) containing 6N sodium hydroxide. Themixture was stirred for 20 minutes, and the reaction solution wasconcentrated and was then purified by preparative chromatography onsilica gel, whereby lepidimoide (1), 2.1 mg, was obtainedquantitatively).

FAB (H₂ O+G) HRFABMS

C₁₂ H₁₇ O₁₀ Na₂ 367.0591 M+Na

[α]_(D) ²¹ +65.2° (c 0.025, D₂ O) Synthetic product

¹ R spectrum: 3,300, 1590 cm⁻¹

¹ H-NMR spectrum: δ (D₂ O)

5.72 (1H, d, J=3.2 Hz), 5.17 (1H, d, J=1.6 Hz), 5.07 (1H, d, J=2.3 Hz),4.26 (1H, dd, J=6.9, 3.2 Hz), 4.08 (1H, dd, J=3.4, 1.6 Hz), 3.79 (1H,dq, J=9.7, 6.8 Hz), 3.76 (1H, dd, J=9.7, 3.4 Hz), 3.72 (1H, dd, J=6.9,2.3 Hz), 3.31 (1H, dd, J=9.7, 9.7 Hz), 1.80 (3H, d, J=6.8 Hz)

(Example 18) Synthesis of compound (13) ##STR25##

3.3 mg in total of compounds (12) and (12') obtained in Example 16 weredissolved in 4 ml methanol. The air in the reaction vessel was degassedand replaced by argon, and 10% palladium-carbon was added at 0° C., andthen the argon in the vessel was replaced at room temperature byhydrogen. The mixture was then stirred for 15 hours at atmosphericpressure. The hydrogen in the vessel was replaced by argon. Afteraddition of Celite, the reaction mixture was filtered. The filtrate wasconcentrated and then purified by preparative silica gel chromatography(chloroform:methanol=50:1), whereby compound (13), 2.9 mg, was obtained(yield: 88%).

(Example 19) Synthesis of compound (14) ##STR26##

2.9 mg of compound (13) obtained in Example 18 was dissolved in acombined solvent of 1 ml water and 1 ml methanol, and an aqueous 6-foldequivalent 1N sodium hydroxide solution was added thereto, and themixture was allowed to react for 3 hours. The reaction solution wasconcentrated and then purified by HP-20 column chromatography, wherebycompound (14), 2.6mg, was obtained (yield: 98%).

¹ H-NMR spectrum (D₂ O): see FIG. 22.

(Test Example 1) Physiological activity of synthetic lepidimoide andlepidimoide derivatives

Compounds (1), (8a), (9a), (10a), (12)+(12'), and (14) synthesized abovewere examined for growth promotion effect on Amaranthus caudatus L.hypocotyls in the same manner as in Example 1 (2) 1.

Amaranthus caudatus L. seeds were placed on a filter paper immersed with0.8 ml of a test solution (an aqueous solution of each compoundsynthesized as described above [10⁻⁵ to 3×10⁻⁴ M]) in a Petri dish of 3cm diameter and were then allowed to stand over 5 days at 25° C. in thedark, and the length of germinated hypocotyls was determined. Thecompound activities expressed in terms of hypocotyl length werecompared.

The results are shown in Table 12.

                  TABLE 12                                                        ______________________________________                                        Comparison of physiological activities of synthetic                           lepidimoide and lepidimoide derivatives                                       Compound No.                                                                         1        8a      9a    10a   12 + 12'                                                                             14                                 ______________________________________                                        Activity                                                                             ++++     ++      ++    ++    +      +++                                ______________________________________                                         ++++: The same as that of natural lepidimoide                                 +++: 75% of the acivity of natural lepidimoide                                ++: 50% of the activity of natural lepidimoide                                +: 25% of the activity of natural lepidimoide                            

What is claimed is:
 1. A compound represented by the formula: ##STR27##wherein R¹, R², R³, R⁴, and R⁵ are the same and each represents ahydrogen atom, an acetyl group or benzyl group, R⁶ represents a hydrogenatom or a hydroxyl group when R¹ through R⁵ are hydrogen atoms, or R⁶represents a benzyloxy group when R¹ through R⁵ are benzyl groups, or R⁶represents a hydrogen atom or an acetoxy group when R¹ through R⁵ areacetyl groups R⁷ represents a hydrogen atom, or R⁶ and R⁷ together mayrepresent the second bond of a double bond, and R⁸ represents a carboxyor methoxycarbonyl group, and salts thereof.
 2. A compound representedby the formula: ##STR28## and salts thereof.
 3. A compound as defined inclaim 1, wherein R¹, R², R³, R⁴, and R⁵ each represent a hydrogen atom,R⁶ represents a hydroxyl group, R⁷ represents a hydrogen atom, and R⁸represents a carboxyl group, and salts thereof.
 4. A compound as definedin claim 1, wherein R¹, R², R³, R⁴, and R⁵ each represent a hydrogenatom, R⁶ represents a hydroxyl group, R⁷ represents a hydrogen atom, andR⁸ represents a methoxycarbonyl group.
 5. A compound as defined in claim1, wherein R¹, R², R³, R⁴, and R⁵ each represent a hydrogen atom, R⁶represents a hydrogen atom, R⁷ represents a hydrogen atom, and R⁸represents a carboxyl group, and salts thereof.
 6. Compounds as definedin claim 1, wherein R¹, R², R³, R⁴, and R⁵ each represent an acetylgroup, R⁶ and R⁷ together represent the second bond of a double bond,and R⁸ represents a methoxycarbonyl group.
 7. A compound as defined inclaim 1 wherein R¹, R², R³, R⁴, and R⁵ each represent a hydrogen atom,R⁶ represents a hydrogen atom or a hydroxyl group, R⁷ represents ahydrogen atom, and R⁸ represents a carboxyl group, and salts thereof.